Comparison of time series clustering methods for identifying novel subphenotypes of patients with infection.

OBJECTIVE: Severe infection can lead to organ dysfunction and sepsis. Identifying subphenotypes of infected patients is essential for personalized management. It is unknown how different time series clustering algorithms compare in identifying these subphenotypes. MATERIALS AND METHODS: Patients with suspected infection admitted between 2014 and 2019 to 4 hospitals in Emory healthcare were included, split into separate training and validation cohorts. Dynamic time warping (DTW) was applied to vital signs from the first 8 h of hospitalization, and hierarchical clustering (DTW-HC) and partition around medoids (DTW-PAM) were used to cluster patients into subphenotypes. DTW-HC, DTW-PAM, and a previously published group-based trajectory model (GBTM) were evaluated for agreement in subphenotype clusters, trajectory patterns, and subphenotype associations with clinical outcomes and treatment responses. RESULTS: There were 12 473 patients in training and 8256 patients in validation cohorts. DTW-HC, DTW-PAM, and GBTM models resulted in 4 consistent vitals trajectory patterns with significant agreement in clustering (71-80% agreement, P < .001): group A was hyperthermic, tachycardic, tachypneic, and hypotensive. Group B was hyperthermic, tachycardic, tachypneic, and hypertensive. Groups C and D had lower temperatures, heart rates, and respiratory rates, with group C normotensive and group D hypotensive. Group A had higher odds ratio of 30-day inpatient mortality (P < .01) and group D had significant mortality benefit from balanced crystalloids compared to saline (P < .01) in all 3 models. DISCUSSION: DTW- and GBTM-based clustering algorithms applied to vital signs in infected patients identified consistent subphenotypes with distinct clinical outcomes and treatment responses. CONCLUSION: Time series clustering with distinct computational approaches demonstrate similar performance and significant agreement in the resulting subphenotypes.

The biphasic and age-dependent impact of klotho on hallmarks of aging and skeletal muscle function.

Aging is accompanied by disrupted information flow, resulting from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders including skeletal muscle wasting, or sarcopenia. To derive a global metric of growing 'disorderliness' of aging muscle, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Escalating network entropy reached an inflection point at old age, while structural and functional alterations progressed into oldest-old age. To probe the potential for restoration of molecular 'order' and reversal of the sarcopenic phenotype, we systemically overexpressed the longevity protein, Klotho, via AAV. Klotho overexpression modulated genes representing all hallmarks of aging in old and oldest-old mice, but pathway enrichment revealed directions of changes were, for many genes, age-dependent. Functional improvements were also age-dependent. Klotho improved strength in old mice, but failed to induce benefits beyond the entropic tipping point.

Regulation of aged skeletal muscle regeneration by circulating extracellular vesicles.

Heterochronic blood exchange (HBE) has demonstrated that circulating factors restore youthful features to aged tissues. However, the systemic mediators of those rejuvenating effects remain poorly defined. We show here that the beneficial effect of young blood on aged muscle regeneration was diminished when serum was depleted of extracellular vesicles (EVs). Whereas EVs from young animals rejuvenate aged cell bioenergetics and skeletal muscle regeneration, aging shifts EV subpopulation heterogeneity and compromises downstream benefits on recipient cells. Machine learning classifiers revealed that aging shifts the nucleic acid, but not protein, fingerprint of circulating EVs. Alterations in sub-population heterogeneity were accompanied by declines in transcript levels of the pro-longevity protein, α-Klotho, and injection of EVs improved muscle regeneration in a Klotho mRNA-dependent manner. These studies demonstrate that EVs play a key role in the rejuvenating effects of HBE and that Klotho transcripts within EVs phenocopy the effects of young serum on aged skeletal muscle.

Caspase1/11 signaling affects muscle regeneration and recovery following ischemia, and can be modulated by chloroquine.

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.

Klotho: An Elephant in Aging Research.

The year 2017 marked the 20th anniversary of the first publication describing Klotho. This single protein was and is remarkable in that its absence in mice conferred an accelerated aging, or progeroid, phenotype with a dramatically shortened life span. On the other hand, genetic overexpression extended both health span and life span by an impressive 30%. Not only has Klotho deficiency been linked to a number of debilitating age-related illnesses but many subsequent reports have lent credence to the idea that Klotho can compress the period of morbidity and extend the life span of both model organisms and humans. This suggests that Klotho functions as an integrator of organ systems, making it both a promising tool for advancing our understanding of the biology of aging and an intriguing target for interventional studies. In this review, we highlight advances in our understanding of Klotho as well as key challenges that have somewhat limited our view, and thus translational potential, of this potent protein.